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Unusual DS-1-like intergenogroup reassortant rotaviruses with a bovine-like G8 genotype (DS-1-like G8P [8] rotaviruses) have emerged and rapidly spread in several countries. In this study, the nucleotide sequences of seven human rotavirus G8P [8] strains in 2017 and 2019 in Japan were determined using viral metagenomics. Its genomic constellation (VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5 genes) was defined as G8-P [8]-I2-R2-C2-M2-A2-N2-T2-E2-H2. Our genetic analysis revealed that the Japanese G8P [8] rotavirus strains in 2017 and 2019 were classified into the same lineages G8-5 and P [8]-3, but they were phylogenetically located on separate branches and belonged to distinct clusters. Our study is the first attempt to investigate the evolution of emerging rotavirus G8P [8] in Japan.

ABSTRACTAlthough two live oral rotavirus (RV) vaccines, Rotarix and RotaTeq, play a critical role toward reducing disease severity, hospitalization, and death rate in RV infections, regular monitoring of vaccine effectiveness (VE) is yet necessary because the segmented genome structure and reassortment capability of RVs pose considerable threats toward waning VE. In this study, we examined the VE by a test-negative study design against G9P[8]I2 strain during a seasonal outbreak in February–May, 2018, in an outpatient clinic in Kyoto Prefecture, Japan. It remains important because G9P[8]I2 strain remains partially heterotypic to these vaccines and predominating in post-vaccination era. During year-long surveillance, RV infections were detected only from February to May. During this outbreak, 33 (42.3%) children out of 78 with acute gastroenteritis (AGE) remained RV-positive, of which 29 (87.8%) children were infected with G9P[8]I2. Two immunochromatographic (IC) assay kits exhibited 100% sensitivity and specificity to detect G9P[8]I2 strain. Only 23.2% children were found to be vaccinated. Yet, significant VE 69.7% (95% CI: 2.5%-90.6%) was recognized against all RV strains that increased with disease severity. Similar significant VE 71.8% (95% CI: 1%-92%) was determined against G9P[8]I2 strain. The severity score remained substantially low in vaccinated children. Our data reveal that vaccine-preventable G9P[8]I2 strain yet may cause outbreak where vaccination coverage remains low. Thus, this study emphasizes the necessity of global introduction of RV-vaccines in national immunization programs of every country.

Noroviruses (NoVs) are the most significant viral pathogens associated with waterborne and foodborne outbreaks of nonbacterial acute gastroenteritis in humans worldwide. This study aimed to investigate the prevalence and diversity of NoVs contaminated in the environmental water in Chiang Mai, Thailand. A total of 600 environmental water samples were collected from ten sampling sites in Chiang Mai from July 2020 to December 2022. The presence of NoV genogroups I (GI), GII, and GIV were examined using real-time RT-PCR assay. The genotype of the virus was determined by nucleotide sequencing and phylogenetic analysis. The results showed that NoV GI and GII were detected at 8.5% (51/600) and 11.7% (70/600) of the samples tested, respectively. However, NoV GIV was not detected in this study. NoV circulated throughout the year, with a higher detection rate during the winter season. Six NoV GI genotypes (GI.1-GI.6) and eight NoV GII genotypes (GII.2, GII.3, GII.7, GII.8, GII.10, GII.13, GII.17, and GII.21) were identified. Among 121 NoV strains detected, GII.17 was the most predominant genotype (24.8%, 30 strains), followed by GII.2 (21.5%, 26 strains), GI.3 (17.4%, 21 strains), and GI.4 (16.5%, 20 strains). Notably, NoV GII.3, GII.7, GII.8, and GII.10 were detected for the first time in water samples in this area. This study provides insight into the occurrence and seasonal pattern of NoV along with novel findings of NoV strains in environmental water in Thailand during the COVID-19 outbreak. Our findings emphasize the importance of further surveillance studies to monitor viral contamination in environmental water.

Mutations in several regions of HCV genome are shown to correlate with response to interferon (IFN) treatment. Persistence of HCV infection and poor susceptibility to treatment might be contributed by mutations arising within HCV genome which enable the virus to escape from host immune response/IFN treatment. This study investigated mutations in core and NS5A genes of HCV from non-responder and relapser patients after treatment with Peg-IFN-α and ribavirin. Viral RNA was extracted from patient sera and core and NS5A genes were amplified by RT-PCR. Nucleotide sequences of the core and NS5A genes were determined by direct sequencing, and converted to amino acid sequences. Nucleotide and amino acid sequences in the core region, ISDR, PKRBD, and V3 regions within NS5A after treatment were highly conserved when comparing to their corresponding sequences obtained before treatment. Interestingly, when comparing the virus from relapsers to those from non-responders, the number of mutations after treatment in N-terminal region of NS5A of virus from relapsers was significantly higher than those from non-responders (P< 0.05). Amino acid mutations at the N-terminus of NS5A of the virus in relapsers might help the virus to survive and somehow relapse after the cessation of the treatment.

Kobuviruses are member of the family Picornaviridae. Initially, members in Kobuvirusgenus were named according to the basis of their host species. The viruses found in humans called “Aichi virus”, the viruses from cattle called “bovine kobuvirus”, and the viruses isolated from pigs called “porcine kobuvirus”. Currently, taxonomy of kobuviruses has been proposed and the virus species have been renamed. The “Aichi virus” has been renamed as “Aichivirus A”, “bovine kobuvirus” has been renamed as “Aichivirus B”, and “porcine kobuvirus” has been changed to “Aichivirus C”. Among Aichivirus A, three distinct members, including Aichi virus 1 (Aichivirus in human), canine kobuvirus 1, and murine kobuvirus 1, have been described. Aichi virus 1 in human is globally distributed and has been identified at low incidence (0–3 %) in sporadic acute gastroenteritis cases. Aichi virus 1 has been reported to be associated with variety types of clinical illnesses including diarrhea, vomiting, fever, purulent conjunctivitis, and respiratory symptoms. The studies from Japan, Spain, Germany, and Tunisia demonstrated that high antibody prevalence against Aichi virus 1 were found in the populations. Aichivirus B or previously known as bovine kobuvirus was first reported in 2003. Since then, Aichivirus B has also been reported from several countries worldwide. An overall prevalence of Aichivirus B varies from 1 to 34.5 %, and the highest prevalence was found in cattle with diarrhea in Korea. Aichivirus C or porcine kobuvirus is widely distributed in pigs. Aichivirus C has been found in both diarrhea and healthy pigs and the positive rate of this virus varies from 3.9 up to 100 %. It was reported that Aichivirus C was found with high prevalence in wild boars in Hungary. The accumulated data of the biological, pathological, as well as epidemiological studies of kobuviruses are still limited. Comprehensive global investigations of the prevalence and diversity are required and will be helpful for providing further insight into pathogenicity, genetic heterogeneity, interspecies transmission, and global distribution of kobuviruses.

Group A rotavirus is the leading cause of severe diarrhea in infants and young children, and in young animals of many species worldwide. Rotavirus is also the major cause of deaths of children younger than 5 years of age, particularly, in developing countries in Asia and Africa. In Thailand, the burden of rotavirus infection rate in children admitted to the hospitals with acute gastroenteritis ranged from 28.4 to 44.5 %. The seasonality of rotavirus gastroenteritis in Thailand was detected all-year-round with the peak from November to April of the following year. The distributions of G genotypes in pediatric patients during twelve-year surveillances of 2000–2011 were G1, G2, G3, G4, G9, and G12. The G9 was detected as the most predominant genotype in 2000–2004 while G1 and G3 were predominated in 2005–2009 and 2009–2011, respectively. The G4 was detected only in 2001–2003 and G12 only in 2007–2009 but was not detectable in any other years of surveillances. For P genotype, P[8] was the only P genotype that always existed as the most predominant with high prevalence. The G–P combination of human rotavirus strains circulated in Thailand were G1P[8], G2P[4], G2P[8], G3P[3], G3P[8], G3P[9], G3P[10], G3P[19], G9P[8], G12P[6], and G12P[8]. The G1P[8] was the most predominant strain followed by G9P[8], G2P[4], G3P[8], G12P[8], G3P[9], G3P[10], G3P[3], G2P[8], G3P[19], and G12P[6]. The studies of animal rotaviruses were performed mainly on porcine rotaviruses and a wide variety of porcine rotavirus strains have been reported, including G2P[27], G3P[6], G3P[13], G3P[19], G3P[23], G4P[6], G4P[13], G4P[19], G4P[23], G5P[6], G5P[13], G9P[7], G9P[13], and G9P[19]. Several unusual strains of human rotaviruses that carried the genes with nucleotide sequences closely related to those of animal rotaviruses have been described in Chiang Mai, Thailand which provided evidences for interspecies transmission of rotaviruses between humans and animals, and also animals to animals are occurring in nature.

ABSTRACTOf 131 stool samples collected from piglets with diarrhea in northern Thailand between July 2006 and August 2008, 14 (10.7%) were positive for group A rotavirus. Sequence analysis showed that 13 strains (92.9%) belonged to the rare P[23] genotype combination with G9 or G3 genotypes.

ABSTRACTA total of 82 fecal specimens which were known to be negative for rotavirus, adenovirus, norovirus, sapovirus, and astrovirus and which were collected from infants and children with acute gastroenteritis in Chiang Mai, Thailand, from January to December 2005 were screened for human parechovirus (HPeV). HPeV was detected by reverse transcription-PCR with a primer pair that amplified the 5' untranslated region of its genome and was genotyped by sequencing of the VP1 region. HPeV was detected in 12 of 82 specimens tested, and the detection rate was found to be 14.6%. The capsid VP1 gene was successfully sequenced from nine of the HPeV strains detected. The HPeV strains studied clustered into four different genotypes, HPeV genotype 1 (HPeV1) to HPeV4, and the majority of the strains studied (five strains) belonged to HPeV1. This is the first finding of HPeV from children with acute gastroenteritis in Thailand. In addition, the diversity of the Thai HPeV strains was also noted.

ABSTRACTEpidemiological surveillance of porcine rotavirus (PoRV) strains was carried out in Chiang Mai Province, Thailand, from 2002 to 2003, and eight rotavirus isolates could not be completely typed by PCR. Of these, six were G3 and one was G4 and displayed a P-nontypeable genotype, while another isolate was both G and P nontypeable. Analysis of a partial VP4 gene of all eight P-nontypeable strains revealed a high degree of amino acid sequence identities (94.7% to 100%), suggesting that they belonged to the same P genotype. Comparison of the amino acid sequences of two representative strains (namely, strains CMP178 and CMP213) with those of 27 other known P genotypes revealed a high degree of amino acid sequence identity with those of P[13] porcine rotavirus reference strains HP113 and HP140, which were recently isolated in India. However, amino acid sequence comparison with non-P[13] rotavirus strains revealed relatively low identities, ranging from 58.2% to 84.8% for full-length VP4 sequences and 35.1% to 80.6% for VP8* sequences. Phylogenetic analysis revealed that CMP178 and CMP213 clustered together in a monophyletic branch with P[13]-like genotypes HP113 and HP140 which was clearly separated from the other lineages of P[13] or P[22] strains. Altogether, these findings indicate that PoRV strains CMP178 and CMP213 should be considered the P[13]-like VP4 genotype, a rare genotype that has been identified only in pigs. This study provides additional evidence of increasing genetic diversity among group A rotaviruses in nature.

ABSTRACTAmong 175 fecal specimens collected from diarrheic piglets during a surveillance of porcine rotavirus (PoRV) strains in Chiang Mai, Thailand, 39 (22.3%) were positive for group A rotaviruses. Of these, 33.3% (13 of 39) belonged to G3P[19], which was a rare P genotype seldom reported. Interestingly, their VP4 nucleotide sequences were most closely related to human P[19] strains (Mc323 and Mc345) isolated in 1989 from the same geographical area where these PoRV strains were isolated. These P[19] PoRV strains were also closely related to another human P[19] strain (RMC321), isolated from India in 1990. The VP4 sequence identities with human P[19] were 95.4% to 97.4%, while those to a porcine P[19] strain (4F) were only 87.6 to 89.1%. Phylogenetic analysis of the VP4 gene revealed that PoRV P[19] strains clustered with human P[19] strains in a monophyletic branch separated from strain 4F. Analysis of the VP7 gene confirmed that these strains belonged to the G3 genotype and shared 97.7% to 98.3% nucleotide identities with other G3 PoRV strains circulating in the regions. This close genetic relationship was also reflected in the phylogenetic analysis of their VP7 genes. Altogether, the findings provided peculiar evidence that supported the porcine origin of VP4 genes of Mc323 and Mc345 human rotaviruses.

An unusual strain of human rotavirus G3P[3] (CMH222), bearing simian‐like VP7and caprine‐like VP4genes, was isolated from a 2‐year‐old child patient during the epidemiological survey of rotavirus in Chiang Mai, Thailand in 2000–2001. The rotavirus strain was characterized by molecular analysis of its VP4, VP6, VP7, and NSP4gene segments. The VP4 sequence of CMH222 shared the greatest homology with those of caprine P[3] (GRV strain) at 90.6% nucleotide and 96.4% amino acid sequence identities. Interestingly, the VP7 sequence revealed highest identity with those of simian G3 rotavirus (RRV strain) at 88% nucleotide and 98.1% amino acid sequence identities. In contrast, percent sequence identities of both the VP4and VP7genes were lower when compared with those of human rotavirus G3P[3] reference strains (Ro1845 and HCR3). Analyses of VP6 and NSP4 sequences showed a close relationship with simian VP6 SG I and caprine NSP4 genotype C, respectively. Phylogenetic analysis of VP4, VP6, VP7, and NSP4genes of CMH222 revealed a common evolutionary lineage with simian and caprine rotavirus strains. These findings strongly suggest multiple interspecies transmission events of rotavirus strains among caprine, simian, and human in nature and provide convincing evidence that evolution of human rotaviruses is tightly intermingled with the evolution of animal rotaviruses. J. Med. Virol. 78:986–994, 2006. © 2006 Wiley‐Liss, Inc.

A total of 402 fecal specimens collected during July 2003–June 2004 from infants and children with acute gastroenteritis, encompassing five localities (Maizuru, Tokyo, Sapporo, Saga, and Osaka) of Japan, were tested for the presence of norovirus by RT‐PCR. It was found that 58 (14.4%) fecal specimens were positive for norovirus. Norovirus infection was detected throughout the year with the highest prevalence in December. Norovirus GII was the most predominant genogroup (98.3%; 57 of 58). The genotypes detected in this study were GI/4, GII/2, GII/3, GII/4, and GII/6. Of these, NoV GII/3 (known as the Arg320 virus cluster) was the most predominant genotype (43.9%), followed by NoV GII/4 (the Lordsdale virus cluster; 35.1%) and others. Two norovirus strains clustered with a “new variant designated GIIb” and a “new variant of GII/4” were found circulating in Japan for the first time. It was interesting to note that NoV GIIb and NoV GII/3 appeared to be the recombinant strains and the recombination site was demonstrated at the overlap of ORF1 and ORF2. The majority (96%) of the dominant norovirus strains were identified as the recombination of GII/3 capsid and GII/12 polymerase. The recombination in the NoV GIIb capsid gene at the breakpoint located at P1 domain was also identified. Obviously, NoV GIIb isolate in Japan had double recombination. This is the first report demonstrating the existence of different “new variants” co‐circulating in Japanese infants and children with acute gastroenteritis. J. Med. Virol. 78:971–978, 2006. © 2006 Wiley‐Liss, Inc.

Among 315 fecal specimens collected from children hospitalized with diarrhea in Chiang Mai, Thailand, in 2000–2001, group A rotavirus was detected in 107 (34.0%). Of these, 98 (91.6%) were G9, 6 (5.6%) were G3 and 3 (2.8%) were G2, respectively. Identification of their P‐types demonstrated that 103 (96.3%) were P[8], 3 (2.8%) were P[4], and 1 (0.9%) was P[3] genotypes. Determination of G‐ and P‐type combination revealed that all of G9 isolates were associated with P[8]. G9P[8] was the most predominant genotype and accounted for the majority (91.6%) of rotaviruses detected in this study. Molecular characterization of these G9 isolates demonstrated that all had long electropherotype, 96 of 98 (98.0%) belonged to subgroup II, one belonged to subgroup I and the other one was subgroup unidentifiable. All of G9 isolates possessed NSP4 genetic group B except for one isolate that showed dual genetic group specificities, B and C. The full‐length VP7gene nucleotide sequences among 15 representatives of these G9 strains were found to be highly homologous with percent identities of 99.3%–100%. Comparison with other G9 strains recently isolated showed that their nucleotide sequences were closely related to those of the US strain, US1205 (98.7%–99.0%) and Thai strain, 97CM108 (98.1%–99.0%). Interestingly, they were most closely related to the Japanese strain, 00‐SG2509VP7, isolated in the same epidemic season, with percent nucleotide sequence identity of 99.4%–99.8%. The data imply that G9 strains isolated in this study and a G9 strain isolated in Japan in the year 2000 might have descended from the same ancestor. J. Med. Virol.78:273–280, 2006. © 2005 Wiley‐Liss, Inc.

A total of 236 fecal specimens collected from infants and children with gastroenteritis in Maizuru city, Japan from July 2002 to June 2003, were tested for the presence of rotaviruses, noroviruses, sapoviruses, astroviruses, and adenoviruses by RT‐PCR, PAGE, RPHA, and latex agglutination methods. Among diarrheal viruses detected, group A rotavirus was the most prevalent (32.2%; 76 of 236) followed by norovirus GII (21.2%; 50 of 236), group C rotavirus (10.2%; 24 of 236), adenovirus (3.8%; 9 of 236), sapovirus (2.5%; 6 of 236), astrovirus (1.3%; 3 of 236), and norovirus GI (0.8%; 2 of 236), respectively. It is noteworthy that group C rotavirus infection was apparently confined only within the period of 5 months (December 2002 through April 2003). This pattern of infection implied that the outbreak of group C rotavirus in these patients, which was the first outbreak of gastroenteritis attributed to group C rotavirus in Maizuru city. Moreover, about half (12 of 24) of group C rotavirus infected cases were confined to infants and young children less than 3 years old. Another interesting feature of the study was the demonstration of the mixed infections with group C rotavirus and group A rotavirus, as well as group C rotavirus and norovirus GII in 20.8% (5 of 24) and 8.3% (2 of 24), respectively. This is the first report of gastroenteritis associated with the mixed infections with group C rotavirus and other viral enteropathogens such as norovirus. The results indicate that group C rotavirus could infect not only older children and adults but also infants and young children under 3 years old. J. Med. Virol. 74:173–179, 2004. © 2004 Wiley‐Liss, Inc.

Fecal specimens from 517 infants and young children admitted to the Civil Karachi Hospital, Dow Medical College, Karachi city, Pakistan with acute gastroenteritis from 1990 to 1994 were collected and screened by RT‐PCR for human astrovirus (AstV), norovirus (NV), and sapovirus (SV). The specific epidemiological data for illness caused by these viruses in Pakistan are not available. AstV, NV, and SV were detected in 58, 51, and 17 of 517 fecal specimens, and this represented 11.2, 9.9, and 3.2%, respectively. An outbreak of gastroenteritis attributable to AstV serotype 1 was identified during September and October 1990. Moreover, one specimen with a triple mixed infection between AstV (serotypes 1 and 3) and NV GII was found. NV and SV were subjected to molecular analysis by sequencing. One of the sequenced specimens positive for SV turned out to be similar to a strain tentatively called a genogroup IV. The result underscores the importance of these viruses in association with acute gastroenteritis in Karachi city, Pakistan. J. Med. Virol. 73:256–261, 2004. © 2004 Wiley‐Liss, Inc.

ABSTRACTWe evaluated a quantitative enzyme immunoassay (trak-C) for hepatitis C virus core antigen (HCV core Ag) by testing serum specimens from 820 injection drug users in Thailand with anti-HCV antibodies. The HCV genotypes in this population include genotypes 3 and 6, which have not been extensively tested with this assay. Among these specimens, 629 (76.7%) yielded positive results, with HCV core Ag concentrations predominantly spanning (35.7%) or above (58.2%) the measurable range of 1.5 to 100 pg/ml. To assess reproducibility, we retested 30 specimens representing six core Ag ranges; the mean coefficient of variation for each range was =9.7% (highest for 1.5 to 25 pg/ml). We also tested 204 specimens of the 820-specimen set for HCV RNA: while 146 (71.6%) were core Ag positive, 168 (82.4%) had detectable HCV RNA, of which 96% were typeable as genotype 3 (39%), 1 (31%), or 6 (26%) by nested reverse transcription-PCR. Among RNA-positive specimens, 86.9% had core Ag; 94% of the RNA negatives were core Ag negative. While there was no apparent bias for detecting core Ag representing the tested genotypes, median quantified results were higher for types 1a and 6 than for genotype 3 (P= 0.01); similarly, the median core Ag concentration was higher in HCV-human immunodeficiency virus-coinfected subjects than in HCV-monoinfected subjects. Our results demonstrated a good correlation between core Ag and HCV RNA in this population with high frequencies of genotypes 3 and 6. Because most core Ag concentrations were greater than those in the measurable range, we recommend a 10-fold dilution of the specimen before quantification. Reproducibility, low technical requirements, and high throughput should make this assay useful for clinical or research monitoring of HCV levels during active infection.

A 4‐year (1996–2000) survey of rotavirus infection involving 2,218 diarrheal fecal specimens of children collected from five regions of Japan was conducted. A total of 642 (28.9%) specimens were found to be rotavirus positive. A changed prevalence pattern of rotavirus G serotype was found with an increase of G9 and G2 and a decrease of G1, although G1 remained the prevailing serotype. Serotype G9 was unexpectedly determined to be the prevailing serotype in Sapporo (62.5%) and Tokyo (52.9%) in 1998–1999, and in Saga (78.4%) in 1999–2000. G9 strains isolated from 1998–1999 belonged to the P[8]‐NSP4‐Wa‐group with long RNA pattern, while, G9 strains isolated from 1999–2000 belonged to three groups, the P[8]‐NSP4‐Wa‐group with long RNA pattern, the P[4]‐NSP4‐KUN‐group with short RNA pattern and a mixed‐type group (P[4]/P[8]‐NSP4‐KUN/Wa‐group with long RNA pattern). Both sequence and immunological analysis of VP7 revealed that the G9 strains from 1999–2000 were much more closely related to the G9 strains isolated worldwide in the 1990s, including G9 strains found in Thailand in 1997. However, the G9 strains from 1998–1999 were distinct from these and more closely related to the G9 prototype strains F45, AU32 and WI61 discovered in Japan and the US in the 1980s. Thus the G9 strains isolated in 1998–1999 had progenitors common to the G9 prototype strains, while the strains isolated in 1999–2000 did not directly evolve from them but were related to global G9 strains that have emerged in recent years. These data supported our previous report that G9 rotavirus might exist as two or more subtypes with diverse RNA patterns, P‐genotype and NSP4 genogroup combinations (Y.M. Zhou et al., J. Med. Virol. 65: 619–628, 2001) and suggested that G9 rotavirus prevalent in Japan during two successive years belonged to different subtypes. The nucleotide sequences presented in this paper were submitted to DDBJ, EMBL and GenBank nucleotide sequence databases. The accession numbers are: 00‐Ad2863VP7 (AB091746), 00‐OS2986VP7 (AB091747), 00‐SG2509VP7 (AB091748), 00‐SG2518VP7 (AB091749), 00‐SG2541 (AB091750), 00‐SG2864 (AB091751), 00‐SP2737VP7 (AB091752), 99‐SP1542VP7 (AB091753), 99‐SP1904VP7 (AB091754), 99‐TK2082VP7 (AB091755) and 99‐TK2091VP7 (AB091756).

Serotyping of human rotavirus was conducted in 396 Japanese and 100 Thai rotavirus‐positive fecal specimens collected from 1995 to 1997. Serotype G9 was found to be the third most common serotype with frequency of 16.2% in Thailand from 1996 to 1997. It was also detected in Japan with a low frequency (0.7%) in this year. The genetic analyses of VP4 and NSP4 genes of these G9 strains showed that 1 strain from Japan possessed P[8] genotype and NSP4 Wa‐group with long electropherotpe (e‐type). In contrast, 5 strains from Thailand belonged to P[6] and 1 strain belonged to P[4]. All of the Thai strains were in the NSP4 KUN‐group with a short e‐type. Sequence analysis of their VP7 gene revealed that there was the highest homology among fecal G9 strains (> 96.3%, amino acid identity) and a relatively high degree of homology to standard viruses, F45 from Japan (95.4–96.3%, amino acid identity) and 116E from India (92–92.3%, amino acid identity). However, immunological analysis using G9 specific monoclonal antibodies (Mabs) against VP7 protein showed that the G9 strains isolated from the two countries had different antigenic specificity. It was confirmed further by intraserotypical phylogenetic analysis of VP7 amino acid. These results indicated that the prevalence of G9 rotavirus in 1996–1997 in Thailand was relative to the continuing recent emergence of it on a worldwide basis, while the Japanese G9 strain isolated in this survey was identified to have progenitors common to the F45 strain that was prevalent in 1985 in Japan. Phylogenetic analysis of VP7 amino acid of G1‐14 prototype rotavirus showed that the G9 strains were most closely related to the equine G14 rotavirus FI23 strain but G3 strains, interserotypically. These findings suggest that G9 rotaviruses might be divided into two or more subtypes. J. Med. Virol. 65:619–628, 2001. © 2001 Wiley‐Liss, Inc.

We performed a placebo-controlled study to evaluate the effects of immunomodulatory treatment with thalidomide on HIV levels, TNF-alpha levels, and immune status of 31 HIV-infected individuals, after temporary suppression of viral replication with antiretroviral drugs. Treatment with a combination of zidovudine and lamivudine (ZDV/LMV) for 14 days resulted in a median decline in plasma viremia of 1.94 log10 RNA equivalents/ml. After discontinuation of ZDV/LMV, thalidomide therapy (200 mg/day for 4 weeks) did not retard the prompt return of HIV titers to the pretreatment levels, and had no effect on plasma levels of TNF-alpha. In contrast, thalidomide treatment resulted in significant immune stimulation. We observed increased levels of plasma soluble IL-2 receptor, soluble CD8 antigen, and IL-12 (p < 0.01 for all parameters), as well as increased cutaneous delayed-type hypersensitivity reactions to recall antigens (p < 0.01) in thalidomide-treated patients. These changes were associated with a median increase in HIV titer of 0.2 log10 RNA equivalents/ml in the thalidomide-treated group (p < 0.05), which resolved after stopping the drug. Further studies were performed in vitro to elucidate the mechanism of thalidomide-induced immune stimulation. When purified T cells from HIV-infected individuals were stimulated by immobilized anti-CD3 in the presence of thalidomide, a costimulatory effect of the drug was observed, resulting in increased production of IL-2 and IFN-gamma, and increased T cell-proliferative responses. Further experiments showed that thalidomide increased IL-12 production by antigen-presenting cells in a T cell-dependent manner. Our findings suggest a potential application for thalidomide as a novel immune adjuvant in HIV disease.

A simple and sensitive procedure of reverse transcriptase polymerase chain reaction (RT‐PCR) was developed previously such that all 4 serotypes of dengue viruses could be detected and their serotypes identified simultaneously in a single‐step procedure. In this study we compared the RT‐PCR with a conventional immunoperoxidase (PAP) staining method for the identification of dengue viruses currently isolated from patient sera. Sixty‐six sera taken from dengue hemorrhagic fever (DHF) patients were subjected to virus isolation by inoculating onto C6/36 cell cultures. Screening for the presence of dengue viruses in culture fluids was done after 7 days of incubation by PAP staining using hyperimmune rabbit anti‐dengue virus antibody as the primary reagent. Dengue viruses in positive cultures were further identified for their serotypes by PAP using type‐specific monoclonal antibodies (MAb) and by RT‐PCR. Thirty‐two out of the 66 serum specimens tested (48.5%) were positive for dengue viruses. Of these, 5 were type 1 (DEN‐1), 25 were type 2 (DEN‐2) and 2 contained both DEN‐1 and DEN‐2. All cultures that were positive by PAP method were also positive by RT‐PCR and vice versa. Thus, the results obtained by RT‐PCR were in good agreement with those by PAP. It is important to point out that while all 5 DEN‐1 isolates reacted readily with the MAb 1F1, only 2 of them could be identified by the MAb 15F3. Our data suggest that antigenic variation among DEN‐1 isolates occur frequently and this should be taken into consideration in the selection of appropriate type‐specific MAb for serotyping of dengue viruses. We also demonstrated dual infection of DEN‐1 and DEN‐2 in two patients' sera by RT‐PCR.

Acute‐ and convalescent‐phase sera from patients with dengue (DEN) hemorrhagic fever (DHF) and Japanese encephalitis (JE) that contained pre‐existing flavivirus antibodies were tested for cross‐reacting antibodies to DEN, JE and yellow fever (YF) viruses by a neutralization (N) test. A fourfold or greater rise in N antibody titer in the convalescent‐phase was considered significant. Of 39 DHF cases, obtained at Chiang Mai University Hospital, Thailand, 15 (38.5%) showed a rise in DEN antibody titer, while another 15 (38.5%) showed a significant rise in both DEN and JE N antibody titers. On the other hand, eight (61.5%) of 13 JE cases obtained at the same Hospital, showed a significant rise in JE antibody titer, while two (15.4%) showed a significant rise in both DEN and JE antibody titers. Sucrose gradient centrifugation and fractionation of these two cross‐reactive JE sera revealed that IgM class antibody was specific for JE, while IgG class antibody was cross‐reactive. Of three JE cases with pre‐existing YF antibody obtained in Okinawa, Japan, two showed a significant rise in YF and JE antibodies. Both IgM and IgG class antibodies to YF virus were elevated. These results indicate that the cross‐reactivity among flaviviruses in different subgroups (complexes), was observed quite often, even by the N test, in sequential flavivirus infection.

ABSTRACTStool specimens from hospitalized infants with sporadic gastroenteritis in Chiang Mai, Thailand, between July 2000 and July 2001 were examined for norovirus and sapovirus by reverse transcription-PCR and sequence analysis. These viruses were identified in 13 of 105 (12%) specimens. One strain was found to be a recombinant norovirus.